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Biochimica et Biophysica Acta 1793 (2009) 625–635

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Biochimica et Biophysica Acta

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Review Proteomics of the

Torben Lübke a, Peter Lobel b,c, David E. Sleat b,c,⁎

a Zentrum Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Georg-August Universität Göttingen, 37073 Göttingen, Germany b Center for Advanced Biotechnology and Medicine, Piscataway, NJ 08854, USA c Department of Pharmacology, University of Medicine and Dentistry of New Jersey - Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA

article info abstract

Article history: Defects in lysosomal function have been associated with numerous monogenic human diseases typically Received 16 May 2008 classified as lysosomal storage diseases. However, there is increasing evidence that lysosomal are Received in revised form 24 September 2008 also involved in more widespread human diseases including cancer and Alzheimer disease. Thus, there is a Accepted 30 September 2008 continuing interest in understanding the cellular functions of the lysosome and an emerging approach to this Available online 15 October 2008 is the identification of its constituent proteins by proteomic analyses. To date, the mammalian lysosome has been shown to contain ∼60 soluble luminal proteins and ∼25 transmembrane proteins. However, recent Keywords: fi fi Lysosomal proteomic studies based upon af nity puri cation of soluble components or subcellular fractionation to Proteomic obtain both soluble and membrane components suggest that there may be many more of both classes of Mass spectrometry protein resident within this organelle than previously appreciated. Discovery of such proteins has important Lysosomal storage disease implications for understanding the function and the dynamics of the lysosome but can also lead the way Mannose-6 phosphate receptor towards the discovery of the genetic basis for human diseases of hitherto unknown etiology. Here, we subcellular fractionation describe current approaches to lysosomal proteomics and data interpretation and review the new lysosomal proteins that have recently emerged from such studies. © 2008 Elsevier B.V. All rights reserved.

1. Introduction to the lysosome very limited tissue distribution and perform specialized cellular functions e.g. the of immune cells [6]. Discovered by Christian de Duve over 50 years ago [1], the In addition to the soluble luminal proteins, many integral and lysosome is a cytoplasmic cellular organelle that has risen to peripheral membrane proteins are associated with the lysosome and prominence because of its critical role in cellular function and tissue have a variety of functions including , transmembrane homeostasis as well as its involvement in numerous human diseases transport of substrates and digestion products, establishment of pH (reviewed in [2,3]). Present in all nucleated eukaryotic cells, the gradients, vesicular transport and maintenance of lysosomal struc- lysosome is delimited by a single-layer lipid membrane and has an tural integrity [3]. acidic internal pH (∼5) that is maintained by an ATP-dependent Classical biochemical and genetic analyses have resulted in the proton pump. The primary cellular function of the lysosome is the characterization of numerous components of the lysosome. More degradation and recycling of macromolecules obtained by endocy- recently, the application of highly-sensitive proteomic approaches has tosis, autophagy and other cellular trafficking pathways. Several lead to the identification of many new proteins that may function in classes of macromolecules are hydrolyzed including proteins, poly- this compartment and for some, lysosomal localization has now been saccharides, lipids and nucleic acids and this is achieved by the verified. It is these new lysosomal proteins that form the focus of this concerted action of numerous soluble catabolic within the review. Approaches to the discovery and validation of lysosomal lumen of the lysosome, collectively termed acid . Acid candidates have recently been reviewed in depth [5] and for the most hydrolases have evolved to function in the low pH of this organelle part, will be outlined in brief here. and possess a wide variety of enzymatic properties. Over 60 of these enzymes and soluble accessory proteins have been described to date. 2. and human disease A number of studies have investigated the proteome of soluble lysosomal proteins from a variety of mammalian tissues and cell types The lysosomal system is of considerable biomedical importance as ([4]; reviewed in [5]) revealing that, for the most part, these proteins alterations in lysosomes and lysosomal proteins are associated with tend to be fairly ubiquitous. However, some lysosomal proteins have numerous human diseases (Table 1) [3,7]. To date, over 50 monogenic human genetic diseases have been identified that are primarily associated with lysosomal dysfunction and the majority of these are fi fi ⁎ Corresponding author. Tel.: +1 732 235 5028. classi ed as lysosomal storage diseases (LSDs). Here, de ciencies in E-mail address: [email protected] (D.E. Sleat). lysosomal proteins, most commonly soluble luminal ones, result in an

0167-4889/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2008.09.018 626 T. Lübke et al. / Biochimica et Biophysica Acta 1793 (2009) 625–635

Table 1 Established lysosomal proteins and associated human disorders

Protein Disease Protein type Protein function Man6-P ABCA2 Membrane Lipid metabolism? ABCB9/TAP-like transporter Membrane Antigen processing ATPase, V-H+ including ∼13 subunits Kufor–Rakeb syndrome/Parkinson disease 9 Membrane Membrane transporter CD68 Membrane CLC-7 Osteopetrosis Membrane Transporter CLN3 protein Ceroid lipofuscinosis, neuronal 3, juvenile Membrane Unknown Cystinosin Cystinosis Membrane Transporter LALP70 Membrane LAPTM4 Membrane LIMP-1/CD63/LAMP-3 Membrane LIMP-2/LGP85 Membrane Lysosomal transport Lipopolysaccharide-induced TNF factor Membrane LITAF LYAAT-1 Membrane Transporter Lysosome-associated membrane Membrane protein 1 (LAMP1) Lysosome-associated membrane Danon disease Membrane Structural, protein import protein 2 (LAMP2) Major facilitator superfamily domain Ceroid lipofuscinosis, neuronal 7, late infantile, Membrane Transporter (?) containing 8 variant Mucolipin Mucolipidosis IV (sialolipidosis) Membrane Membrane transporter Nicastrin Membrane NPC1 protein Niemann-pick disease, type C1 Membrane Membrane transporter (?) Solute carrier family 17 Sialuria (Salla disease) Membrane Membrane transporter 1-O-acylceramide synthase Soluble Catabolic + Acid ceramidase Farber disease Soluble Catabolic enzyme + Acid Wolman disease Soluble Catabolic enzyme + Alpha-galactosidase A Fabry disease Soluble Catabolic enzyme + Alpha-L-iduronidase Mucopolysaccharidosis type I (Hurler and Scheie Soluble Catabolic enzyme + syndromes) Alpha-N-acetylgalactosaminidase Schindler disease, type I Soluble Catabolic enzyme + Alpha-N-acetylglucosaminidase Mucopolysaccharidosis type IIIb (Sanfilippo Soluble Catabolic enzyme + syndrome B) A Metachromatic leukodystrophy Soluble Catabolic enzyme + Mucopolysaccharidosis type VI Soluble Catabolic enzyme + (Maroteaux-Lamy syndrome) Beta-galactosidase Mucopolysaccharidosis type IVb (Morquio Soluble Catabolic enzyme + syndrome B) Beta-glucuronidase Mucopolysaccharidosis type VII (Sly syndrome) Soluble Catabolic enzyme + Beta- alpha chain Tay–Sachs disease Soluble Catabolic enzyme + Beta-hexosaminidase beta chain Sandhoff disease Soluble Catabolic enzyme + Beta-mannosidase Mannosidosis, beta A, lysosomal Soluble Catabolic enzyme + , vitellogenic-like Soluble Catabolic enzyme ? + B Soluble Catabolic enzyme + Cathepsin D Ceroid lipofuscinosis, neuronal 10, congenital Soluble Catabolic enzyme + Cathepsin F Soluble Catabolic enzyme + Cathepsin H Soluble Catabolic enzyme + Cathepsin K Pycnodysostosis Soluble Catabolic enzyme + Cathepsin L Soluble Catabolic enzyme + Cathepsin O Soluble Catabolic enzyme + Cathepsin S Soluble Catabolic enzyme + Cathepsin Z Soluble Catabolic enzyme + CLN5 protein Ceroid lipofuscinosis, neuronal 5, late infantile, Soluble Unknown + variant II Soluble Catabolic enzyme + Dipeptidyl-peptidase I Papillon-Lefevre syndrome Soluble Catabolic enzyme + Galactocerebrosidase Krabbe disease Soluble Catabolic enzyme + Gamma-glutamyl Soluble Catabolic enzyme + Glycosylasparaginase Aspartylglucosaminuria Soluble Catabolic enzyme + GM2 activator Tay–Sachs disease, AB variant Soluble Accessory protein + Hyaluronidase Mucopolysaccharidosis type IX (hyaluronidase Soluble Catabolic enzyme + deficiency) Iduronate 2- Mucopolysaccharidosis type II (Hunter Soluble Catabolic enzyme + syndrome) Interferon gamma inducible protein 30 Soluble Catabolic enzyme + Legumain Soluble Catabolic enzyme + Lysosomal alpha-glucosidase Glycogen storage disease II (Pompe disease) Soluble Catabolic enzyme + Lysosomal alpha-mannosidase Mannosidosis, alpha b, lysosomal Soluble Catabolic enzyme + Lysosomal protective protein/Cathepsin A deficiency with beta- Soluble Catabolic enzyme/accessory protein + galactosidase deficiency Myeloperoxidase Myeloperoxidase deficiency Soluble Host defence + N-acetylgalactosamine-6-sulfatase Mucopolysaccharidosis type IVa (Morquio Soluble Catabolic enzyme + syndrome a) N-acetylglucosamine-6-sulfatase Mucopolysaccharidosis type IIId (Sanfilippo Soluble Catabolic enzyme + syndrome d) NPC2 protein Niemann-pick disease, type C2 Soluble Soluble transporter + T. Lübke et al. / Biochimica et Biophysica Acta 1793 (2009) 625–635 627

Table 1 (continued) Protein Disease Protein type Protein function Man6-P N-sulphoglucosamine sulphohydrolase/ Mucopolysaccharidosis type IIIa (Sanfilippo Soluble Catabolic enzyme + heparan N-sulfatase syndrome a) Palmitoyl-protein 1 Ceroid lipofuscinosis, neuronal 1, infantile Soluble Catabolic enzyme + Palmitoyl-protein thioesterase 2 Soluble Catabolic enzyme + Saposin Combined saposin deficiency; Krabbe disease, Soluble Accessory protein + atypical, due to saposin a deficiency; metachromatic leukodystrophy due to saposin B deficiency; Gaucher disease, atypical, due to saposin C deficiency Sialic acid 9-O- Soluble + Sialidase 1 Mucolipidosis I (sialidosis) Soluble Catabolic enzyme + Sialidase 4 Soluble Catabolic enzyme + Sphingomyelin Niemann-Pick disease, type a and b Soluble Catabolic enzyme + Tartrate-resistant acid phospahatase Soluble Catabolic enzyme + Tissue alpha-L-fucosidase Fucosidosis Soluble Catabolic enzyme + Tripeptidyl-peptidase I Ceroid lipofuscinosis, neuronal 2, late infantile Soluble Catabolic enzyme + Lysosomal acid Soluble, membrane associated Catabolic enzyme – Gaucher disease Soluble, membrane associated Catabolic enzyme –

Many membrane proteins involved in cellular trafficking have multiple cellular locations and some may be transiently associated with the lysosome (e.g., recycling receptors and adaptors involved in vesicular targeting). We have therefore limited this list to membrane proteins that appear to be primarily located to the lysosome and which function within this organelle.

accumulation of storage material within the lysosome. Storage unknown genetic etiology that appear to be lysosomal in origin material primarily represents undigested substrates and may result based upon morphological or other clinical criteria. For example, the in alterations in the ultrastructure of the lysosome that are often presence of membrane-delimited storage bodies, especially when specific to a given LSD and which may be diagnostically valuable. associated with progressive neurodegeneration, would be highly Defects in various classes of lysosomal proteins result in disease suggestive of a lysosomal storage disease. There are a number of (reviewed in [7]) but most LSDs result from in clinically defined diseases that fall into this category, notable encoding lysosomal enzymes. Other LSDs result from defects in examples of which are the adult form of neuronal ceroid lipofuscinosis soluble accessory proteins that are involved in functions such as and geleophysic dysplasia (reviewed in [5]). presenting substrates to hydrolytic enzymes, membrane proteins that To date, proteomic approaches have led to the identification of transport degradation products out of the lysosome or components of three human lysosomal disease genes. In a comparative proteomic the cellular machinery involved in the processing or trafficking of study [14], lysosomal proteins in late infantile neuronal ceroid lysosomal proteins. Individually, LSDs are rare but as a group, their lipofuscinosis, a fatal neurodegenerative disease of children, were frequency is significant, with an incidence of ∼1:5000 live births [8]. compared to those in normal controls. A previously uncharacterized In addition to these diseases, there is increasing evidence that lysosomal protein was found to be absent in the patient samples and lysosomes and lysosomal activities may be involved in more wide- mutations were subsequently found in its respective , con- spread, polygenic diseases such as cancer [9], arthritis [10], athero- firming it to be the basis for disease. This protein was eventually sclerosis [11] and Alzheimer's disease [12]. Of particular recent demonstrated to be I, a lysosomal interest is the function of the lysosome in cellular autophagic [15,16]. pathways that may play a protective role against infection, aging, Two other proteomic studies led to the discovery of disease genes neurodegeneration and cancer (reviewed in [13]). not by a comparative approach but by instead providing candidates for further genetic analysis. In a study of purified soluble lysosomal 3. Significance of lysosomal proteomic studies proteins, a previously characterized cholesterol binding protein was found and confirmed to reside within the lysosome [17] and this As interest in the role of the lysosomal system under normal information provided a rationale for a genetic screen of this protein in conditions and in human disease continues to increase, more studies unsolved lysosomal storage diseases exhibiting cholesterol storage. are being directed towards the elucidation of the mammalian Mutations were found in the gene encoding this protein in Niemann- lysosomal proteome and that of related organelles (reviewed in Pick C Type 2, a neurovisceral disease of children that was of un- [3,5]). Understanding the full range of biological functions of this known basis at the time. Mucopolysaccharidosis IIIC (MPSIIIC) is a organelle provides essential information regarding the normal lysosomal disease that was known to be caused by a loss of heparan function of the cell but may also shed insights into how lysosomes acetyl-CoA:alpha-glucosaminide N-acetyltransferase (HGSNAT) may influence general cellular activities in pathogenic states. In some activity and while the gene encoding this protein had been mapped cases, changes in lysosomal activities may be directly involved in by linkage analysis, it had not been identified [18]. In a proteomic disease and thus identifying such changes could potentially lead to study of purified lysosomal membrane proteins [19],anovel novel therapeutic targets. Other alterations in lysosomal activities membrane protein was identified whose gene mapped to the same may not be directly involved in pathogenesis per se but may reflect as that determined for the defect in MPSIIIC. Subsequent disease and could potentially be useful as prognostic or diagnostic analysis of this novel lysosomal protein revealed mutations in MPSIIIC markers. It is worth noting that while select lysosomal activities can and confirmed that it encoded HGSNAT [20]. be individually measured in disease samples, the application of In addition to clinically-defined diseases of unknown basis, there quantitative proteomic approaches can provide a global picture of the are numerous patients diagnosed with apparent lysosomal storage lysosomal system in particular by revealing coordinate alterations in disease in which the gene defect cannot be found. Many of these expression. cases may represent atypical clinical presentation of established Another rationale for the comprehensive characterization of the LSDs, often with a mild or delayed onset resulting from partial loss of mammalian lysosome is that there remain human diseases of function mutations [21]. However, some cases are likely to result 628 T. Lübke et al. / Biochimica et Biophysica Acta 1793 (2009) 625–635 from defects in lysosomal proteins that are not currently associated were prepared from human placenta by Percoll gradient centrifuga- with human disease while others might be caused by mutations in tion and constituent proteins identified by N-terminal sequencing by genes encoding lysosomal proteins that have not yet been identified Edman degradation after fractionation by two-dimensional gel or assigned to this organelle. In all of these cases, lysosomal pro- electrophoresis, resulting in the assignment of eight lysosomal teomic analyses could play a pivotal role in the identification of gene proteins [30]. In another study, rat liver tritosomes prepared by defects. sucrose density gradient centrifugation were analyzed using mass spectrometric methods for protein identification and 215 proteins 4. Organellar proteomics, subcellular fractionation and were confidently identified [19]. Some of these proteins were application to the lysosome previously designated to be lysosomal but the majority were assigned to other organelles. While some cellular proteins certainly have Proteomic characterization of individual organelles [22] can multiple cellular locations, this does illustrate the problems of false provide valuable information regarding their function in normal and positive localization (discussed earlier) that emerge in the analysis of disease states. In addition, this type of approach circumvents organelle-enriched fractions. Methods to address this problem are analytical issues associated with the complexity of the entire discussed below (Section 6). proteome and represents a tractable method for determining the Protocols have also been established to isolate lysosomes by proteome of a given tissue or cell type. The most widely applied selectively loading with dextran [31], iron [32] or gold [33]. These approach towards organellar proteomics involves subcellular fractio- different approaches undoubtedly have individual merits but, to date, nation, typically by differential and gradient centrifugation, and none have been applied to lysosomal proteomics studies. protein identification using liquid chromatography tandem mass spectrometry (LC-MS/MS) and these methods can be applied towards 5. Affinity purification of lysosomal proteins the lysosomal proteome. However, because of the overlapping physical properties of organelles, the resolution of subcellular A subgroup of lysosomal proteins representing the majority of fractionation by density centrifugation is limited, typically allowing soluble luminal components can be purified by affinity chromato- for the enrichment of organelles but not purification to homogeneity. graphy for proteomic characterization. Newly synthesized lysosomal As a result, these experiments are highly susceptible to false positive proteins contain a specific carbohydrate modification, mannose 6- errors in assignment of location and data must be interpreted carefully phosphate (Man6-P) that is recognized and bound by two Man6-P with appropriate validation. This problem is compounded by the fact receptors (MPRs). MPRs direct the targeting of lysosomal proteins that some proteins may be present in multiple cellular locations. For from the trans-Golgi to an acidified prelysosomal compartment where example, proteins derived from mitochondria may be found in both the Man6-P modification is variably removed depending on tissue and mitochondria and lysosomes involved in autophagic degradation of cell type. Purified MPRs can be used for the visualization of Man6-P mitochondria. Lysosomal proteins may be found in lysosomes as well glycoproteins in biological samples [34]. In addition, when immobi- as their sites of synthesis (ER and Golgi) and in transport vesicles. lized on a solid support, purified MPRs can be used for the affinity There are emerging methodological improvements that increase purification of Man6-P containing proteins which are specifically the accuracy of analytical or preparative subcellular fractionation eluted from the immobilized MPR by free Man6-P, an approach first approaches to organellar proteomics. In a density gradient, organelles described in an analysis of Man6-P glycoproteins purified from rat migrate with overlapping but distinct profiles thus, rather than simply brain [35]. characterizing the proteome of organelle-enriched preparations, one Early proteomic analyses [35,36] of purified Man6-P glycoproteins approach is to use quantitative mass spectrometric methods to relied upon N-terminal sequencing for protein identification but measure the distribution of proteins across the gradient and assign subsequent studies have employed more sensitive mass spectrometric location by reference to the distribution of marker proteins. Protein methods and have focussed on a wide variety of sources of these distribution can be measured using quantitative isotopic labeling [23] proteins. Proteins containing Man6-P have now been affinity-purified or label-free methods [24]. from 17 different rat tissues [4,35], mouse brain [37] and plasma [38], Another approach for definitive localization is to use methods that human brain [37,39], plasma [40] and urine [36,41]. The same specifically alter the biophysical properties of organelles, resulting in a approach has also been used to purify Man6-P glycoproteins from characteristic shift of their density as measured by gradient centrifu- media conditioned by mouse osteoclasts that naturally secrete gation. The distribution of marker enzymes for a given organelle is lysosomal proteins [42], human monocytes and breast cancer cell thus altered and other proteins with a similar shift in distribution can lines that were chemically induced to secrete lysosomal proteins thereby be assigned to this compartment. For the lysosome, a selective [43,44] or mouse embryonic fibroblasts that secrete lysosomal shift in buoyant density can be achieved in a number of ways. proteins in the absence of both MPRs [45]. Taken together, proteomic Treatment of animals with Triton WR-1339 (tyloxapol) prior to tissue analysis of purified Man6-P glycoproteins has resulted in the harvest and subcellular fractionation causes a lysosomal accumulation identification of 60 known lysosomal proteins as well as numerous of lipids in the so-called “tritosomes” [25,26]. Loss of NPC2, a candidates that may also have lysosomal function (reviewed in [5]). lysosomal cholesterol binding protein, results in an accumulation of Other classes of proteins are also identified in these studies including cholesterol and other lipids within the lysosome with a concomitant contaminants that are not specifically eluted from the immobilized MPR decrease in density [27]. In addition, treatment of cultured cells with (i.e., “sticky” or abundant proteins). In addition, some non-lysosomal progesterone also results in a shift in the buoyant density of lysosomes proteins, e.g., lectins and protease inhibitors, may bind and copurify [28], presumably by blocking cholesterol egress [29]. For each of these with true Man6-P glycoproteins. One approach that has proven useful methods, the density of other organelles remains largely unchanged, to differentiate between true Man6-P glycoproteins and other pro- thus the shift in distribution represents a specific test for lysosomal teins has been to directly identify the site of the N-linked oligosaccha- localization. In the past, such approaches have been limited to the ride that contains Man6-P using mass spectrometric methods after analysis of proteins for which biochemical tests or immunological deglycosylation of purified Man6-P containing glycopeptides with reagents are available. However, the use of mass spectrometry for Endoglycosidase H [37].Thismethodhasverified the presence of protein identification and quantitation in subcellular fractionation will Man6-P in numerous proteins not previously thought to contain this greatly expand the application of these methods. modification. However, based on the identification of proteins known to A number of studies have used subcellular fractionation to contain Man6-P, a significant drawback of this approach is that not all investigate the lysosomal proteome. In an early study, lysosomes deglycosylated peptides that are known to be present are assigned and T. Lübke et al. / Biochimica et Biophysica Acta 1793 (2009) 625–635 629 therefore proteome coverage appears to be limited. Most likely, this proteins in all subcellular and extracellular fractions, not just the simply reflects the specific properties (e.g. sequence and size) of a given lysosome. peptide that may cause it to be selectively lost during sample For preparations of affinity purified Man6-P glycoproteins, a recent preparation or to be missed by the mass spectrometer because it is approach to exclude contaminants is based upon a comparison poorly ionized or it is outside the mass range constraints of the between mock and specific eluates of the immobilized MPR affinity instrument. Alternatively, failure to detect a given Man-6-phosphoryla- columns [4]. After loading and washing, columns are first eluted with tion site may simply reflect low peptide abundance. buffer containing mannose and glucose 6-phosphate (the “mock” It is worth noting that while the majority of soluble lysosomal eluate) then eluted with buffer containing Man6-P (the specific proteins are targeted to the lysosome via the Man6P-dependent eluate). Spectral counting was then used to estimate the relative pathway, other pathways do exist thus there may be multiple ways abundance of each identified protein in both eluates with the in which a given lysosomal protein can reach its final destination. prediction that true Man6-P glycoproteins (but possibly also proteins Some soluble lysosomal proteins are targeted solely by Man6P- that do not contain Man6-P but which associate with Man6-P independent routes and will not be affinity purified on immobilized glycoproteins, depending upon the strength of interaction) should MPR. An example is β-glucocerebrosidase which is targeted to the be enriched in the Man6-P eluate relative to the mannose/glucose 6- lysosome via interaction with the lysosomal transmembrane protein phosphate eluate. In contrast, non-specific contaminants (i.e., abun- LIMP2 [46]. dant or “sticky” proteins that leach from the column in a Man6-P independent manner) should be present at equal or greater levels in 6. Approaches to distinguish candidate novel lysosomal proteins the mock compared to specific eluate. Based upon known lysosomal from contaminants proteins, this prediction proved to be the case, with 59/60 of these proteins with the lower 95% confidence interval of the enrichment In the characterization of lysosomal proteomes prepared by both ratio being greater than 2.75 [4]. Spectral count analysis revealed 52 subcellular fractionation and affinity purification, distinguishing proteins that are not currently assigned to the lysosome that were candidates for novel lysosomal proteins from contaminants and similarly enriched and thus represent primary candidates for other classes of protein remains problematic. To some extent, lysosomal localization. In concept, the approach of comparing protein knowledge-based interpretation of data can be helpful; for example, levels in mock versus specificaffinity column eluates is similar to the I- homologs of known lysosomal proteins are particularly likely to reside DIRT procedure for identifying specific members of a protein complex within this organelle. An example is acid-sphingomyelinase like 3A, that are isolated by the affinity tagging of one of its constituents [49]. which has been found in numerous subcellular fractionation and However, one important difference is that estimation of protein levels affinity purification studies and which is almost certainly a true in the MPR affinity column eluates was achieved by spectral counting lysosomal protein. However, this sort of analysis fails with proteins rather than isotopic labelling. This decreases the number of experi- where a known or predicted function remains to be determined. An mental manipulations and simplifies data analysis but there are example of such a protein is mammalian ependymin-related protein 1, limitations to this approach. which is lysosomal [27,45] but of completely unknown function. Given Inherent to the use of spectral counting as a tool in assigning that many of the most interesting new lysosomal proteins will be lysosomal localization in experiments based upon subcellular fractio- those of novel function, better methods to differentiate between nation or affinity chromatography is that the confidence of the candidates and other proteins are needed. conclusions are highly dependent on the number of spectra observed. Methods based upon quantitative LC-MS/MS are now beginning to Statistical theory allows calculation of confidence intervals for the emerge that can help with this challenging problem. Schröder et al. relative abundance ratio of a protein in two samples of interest [4,38]. [47] have recently reported an analysis of lysosomal membrane The 95% confidence intervals are quite wide for proteins with low proteins isolated from human placenta and while the preparative spectral counts and, as a result, significant conclusions are frequently approaches were conventional, this study differs from previous difficult to make with respect to less abundant proteins in a mixture. analysis of similar samples in that a statistical analysis was Targeted mass spectrometric analyses that limit sampling of abundant incorporated to exclude contaminants [47]. In this approach, a species and promote sampling of minor species may help. However, lysosomal “dense pool” fraction which contained lysosomes, mito- measurement of protein abundance using isotope labeling methods is chondria and other material, was first prepared from placenta by much less dependent on the number of spectra assigned to an Percoll gradient centrifugation. Lysosomes were disrupted by incuba- individual protein and may be especially useful in investigating low tion with methyl ester, which is hydrolyzed within the abundance proteins. These approaches or others quantitative meth- lysosome resulting in their selective disruption via osmotic stress. ods, e.g. peptide peak integration combined with high-resolution MS, Mitochondria were then removed by sucrose density gradient are likely to have increasingly significant application in the investiga- centrifugation and lipofuscin removed by a further Percoll gradient, tion of lysosomal proteomes. resulting in an apparently homogeneous lysosomal membrane preparation. The lysosomal membrane and the dense pool samples 7. Approaches to the validation of lysosomal candidates were then analyzed by LC-MS/MS and enrichment in the former determined by spectral counting, a semiquantitative method for Novel lysosomal candidates can be identified by MPR affinity measuring relative protein abundance [48]. Based upon known purification or by subcellular fractionation but for either approach, lysosomal proteins, a threshold for enrichment was established and there remains a need for the validation of cellular location. Most this was used to filter the data set together with a threshold for the proteins containing Man6-P reside within the lysosome but there are probability of enrichment. This resulted in exclusion of the majority of some that contain this modification that do not reside within this known mitochondrial, ER, plasma membrane and peroxisomal organelle or which may do so only transiently (e.g., glycoproteins that proteins identified and allowed for the most accurate description of are aberrantly phosphorylated and thus targeted to the lysosome the lysosomal membrane proteome to date. However, while this study where they are rapidly degraded). Proteins assigned to the lysosomal certainly represents a significant step forward in subcellular fractio- compartment by subcellular fractionation could represent new nation-based lysosomal proteomics, one limitation is that lysosomal lysosomal proteins or alternatively, they could be contaminants constituents cannot be distinguished from proteins undergoing from other organelles, proteins undergoing degradation in the degradation within the lysosomal compartment. In order to achieve lysosome or proteins that associate with the lysosomal membrane this, it will be necessary to follow the total distribution of candidate after cell lysis. 630 T. Lübke et al. / Biochimica et Biophysica Acta 1793 (2009) 625–635

There are two primary ways of achieving this, each with their own dependent manner and has been directly demonstrated to contain particular merits and limitations (reviewed in [5]). First, morpholo- Man6-P residues [37,45,55]. Endogenous CREG1 did not co-localize gical approaches to compare the cellular distribution with lysosomal with LAMP-1 [55] whereas other studies [45] could show that after markers can provide valuable information. Endogenous or recombi- internalization in human fibroblasts, a C-terminal tagged version of nant expressed proteins can be detected immunohistochemically or CREG1 localizes to LAMP1-positive lysosomes. In addition, in by tagging with a recombinant epitope or fluorescent protein but subcellular fractionation analyses of rat liver, CREG1 was found to there are drawbacks: generation and validation of antibodies can be codistribute with lysosomal markers in untreated rats and rats treated difficult and time-consuming for previously uncharacterized proteins; with Triton WR-1339 (M. Qian and Lobel, unpublished data) (see immuno- or fluorescent tags are prone to degradation in the protease Section 4). Recently, a lysosomal localization for CREG1 was unequi- rich environment of the lysosome [45,50]; and expression of vocally demonstrated by subcellular fractionation and immunoloca- lysosomal proteins at supraphysiological levels can result in incorrect lization [57] although its function within this organelle remains to be intracellular targeting or secretion from the cell [51,52], complicating elucidated. assignment of cellular location. Second, analytical subcellular fractio- nation can provide a powerful tool in determining the subcellular 8.3. Arylsulfatase G (ARSG) localization of a protein of interest, especially when combined with methods that elicit a specific shift in lysosome density as discussed Initially identified by a bioinformatic search of EST databases earlier. Again, however, antibody reagents may be required for those [58], ARSG is a novel sulfatase gene with significant similarity to proteins that lack a functional assay. lysosomal (37% identical, 50% similar). Initial studies of recombinant ARSG expressed in COS-7 cells suggested that it was 8. Novel lysosomal M6P-containing proteins localized to the ER [58] but the isolation of this protein by MPR- affinity purification and verification of Man6-phosphorylation Increasingly sensitive mass spectrometry techniques for protein strongly suggested lysosomal localization. A subsequent study also identification have resulted in the identification of a large number of showed that this protein binds MPR and importantly, confirmed a proteins from numerous sources when purified by affinity chroma- lysosomal localization by immunohistochemistry [59].Inthesame tography on immobilized MPRs. These proteins can be classified into study, ARSG was also demonstrated to have sulfatase activity at a broad range of categories based upon their known or predicted acidic pH. properties: 1) known lysosomal Man6-P glycoproteins; 2) poten- tially new lysosomal proteins; 3) proteins assigned to cellular com- 8.4. Arylsulfatase K, telethon sulfatase (ARSK) partments other than the lysosome and 4); probable contaminants. Here, we will summarize the current state of knowledge regarding Another member of the arylsulfatase family, ARSK, was also some potentially new lysosomal proteins and also discuss a number identified by bioinformatic analyses [60] and later identified by of proteins that have been demonstrated to contain Man6-P but MPR-affinity purification and shown to contain Man6-P. ARSK is a 536 which have been assigned to other cellular or extracellular loca- amino acid protein with relatively little homology with other tions. For consistency, human is used even (up to 22% identity) and such an activity has yet to be though for some proteins, orthologs in mouse and rat were originally demonstrated experimentally. The precise spatial orientation of key identified. amino acids are essential for sulfatase function and these are The following proteins have been identified in proteomic analyses conserved in ARSK, consistent with a sulfatase activity [61]. of purified Man6-P glycoprotein preparation and may represent new lysosomal proteins. Table 2 summarizes the reports in which each 8.5. N-acylsphingosine amidohydrolase (acid ceramidase)-like (NAAA) protein was identified and summarizes evidence for lysosomal localization including whether the presence of Man6-P has been Isolated by MPR-affinity purification from mouse brain, NAAA is directly verified using mass spectrometric methods [37]. a 359 amino acid N-acylethanolamine-hydrolyzing acid amidase that is related (33% identical, 52% similar) to the established 8.1. -like phosphodiesterase 3a (SMPLD3a) lysosomal acid ceramidase. NAAA was first purified from human megakaryoblastic cells [62] and subsequently found in various rat SMPDL3a was first identified as an interacting partner of the tumor tissues [63]. GFP-tagged NAAA derivatives were originally suggested suppressor gene DBC1 (formerly DBCCR1) in a yeast 2-hybrid screen to localize within the lysosome [64,65] and recently, this has been [53]. The biological significance of this potential interaction is not demonstrated for the endogenous protein [66]. NAAA was originally known and remains to be corroborated by other approaches. The found to hydrolyze anadamide at acidic pH [62] and later shown to cellular function of SMPDL3a is not known. The eponym sphingo- hydrolyze bioactive N-acylethanolamines into free fatty acids and myelinase-like is inferred from its homology (30% identical, 47% ethanolamine [65]. similar) to lysosomal acid sphingomyelinase but such an activity has not yet been demonstrated by biochemical means. 8.6. Mannose-6-phosphate protein P76 (P76, LOC196463)

8.2. Cellular repressor of E1A-stimulated genes (CREG1) P76 (formerly hypothetical protein LOC196463) was first identified as a relatively abundant constituent of preparations of Man6-P CREG1 was first described as a cellular protein with some sequence glycoproteins from humans and rodents and has now been convin- similarity to the adenoviral E1A oncogene that was proposed to play a cingly demonstrated to be located within the lysosome [45,67,68]. role in transcriptional regulation of cell growth and differentiation Recombinant P76 was internalized and delivered to the lysosome of [54]. CREG1 is a 220 amino acid glycoprotein that is secreted when cultured cells in a MPR-dependent manner [45] and, consistent with overexpressed and which has been also suggested to function this observation, carbohydrate residues containing Man6-P were intracellularly as a transcriptional repressor by counteracting E1A in identified at 5 of 6 potential N-linked sites of P76 human teratocarcinoma cells [55]. CREG1 has also been suggested to isolated from human brain [37]. It is synthesized as single chain promote differentiation and cell growth arrest by the inhibition of precursor that is processed to multiple smaller chains [37,45,67,68] ERK1/2 in cultured vascular smooth muscle cells [56]. Secreted CREG1 The function of P76 is not known. This protein has significant binds to the MPR300 (CI-MPR; M6P/IGF2R) in a glycosylation- homology (36% identical, 52% similar) with B T. Lübke et al. / Biochimica et Biophysica Acta 1793 (2009) 625–635 631

Table 2 Potentially novel soluble lysosomal proteins that were identified in Man6-P glycoproteomic analyses

Protein Gene Ref. M6P sites verified Lysosomal validation AOAH b, c Yes Arylsulfatase G ARSG h Yes Arylsulfatase K ARSK c, h, i Yes N-acylsphingosine amidohydrolase 2 ASAH2 h N-acylethanolamine-hydrolyzing acid amidase NAAA h, i Biotinidase precursor BTD c, f, h, i Yes Cat eye syndrome critical region 1 CECR1 c, f, g Yes Clusterin CLU c, f, h, i Cellular repressor of E1A-stimulated genes CREG1 a, b, c, d, f, i Yes Morphological, subcellular fractionation⁎ Deoxyribonuclease 1 DNASE1 g, i Mammalian ependymin related protein 1 EPDR1 c, d, f, h, i Yes Morphological, subcellular fractionation Epididymis-specific alpha-D-mannosidase MAN2B2 a, c, d, e, f, g, h, i Yes ER 1 ERAP1 f, i Hypothetical protein FLJ22662 FLJ22662 c, f, g, i Yes Plasma alpha-L-2-fucosidase FUCA2 c, f, i Yes Interleukin-4-induced gene 1 IL4I1 e, i Morphological Mannose-6-phosphate protein P76 LOC196463 c, d, f, g, h, i Yes Morphological, subcellular fractionation Phospholipase D3 PLD3 c, h, i Procollagen- 1,2-oxoglutarate 5-dioxygenase 1 PLOD1 b, e, f, g, i Protein O-fucosyltransferase 1 POFUT1 h, i Protein O-fucosyltransferase 2 POFUT2 c, f, h, i Yes Prostaglandin-H2 D- PTGDS c, f, h Yes Pancreatic RNASE1 g, f Ribonuclease 6 RNASE6 d, f, g, i RNASET2 c, e, f, g, h, i Subcellular fractionation Serine carboxypeptidase 1 SCPEP1 c, d, e, h, i Yes Morphological, subcellular fractionation⁎ Neuroserpin SERPINI1 c, i Yes Acid sphingomyelinase-like phosphodiesterase 3A SMPDL3A c, e, f, g, h, i Yes Microsomal stress 70 protein ATPase STCH c, f, g, i Yes Sulfatase modifying factor 2 SUMF2 c, f, i Yes

References are: a, Journet et al. [43], Electrophoresis 21, 3411–3419; b, Journet et al. [44], Proteomics 2, 1026–1040; c, Sleat et al. [39], Proteomics 5, 1520–1532; d, Kollmann et al. [45], Proteomics 5 3966–3978; e, Czupalla et al. [42], Mol Cell Proteomics 5, 134–143; f, Sleat et al. [37], Mol Cell Proteomics 5, 1942–1956.; g, Sleat et al. [5], Biochim Biophys Acta 1774, 368–372 ; h, Qian et al. [35], Mol Cell Proteomics 7, 58–70; and i, Sleat et al. [4], J Prot Research 7, 3010–3021.

() from Dictyostelium discoideum, therefore a func- EPDR1 contains at least one carbohydrate with Man6-P and has been tion in lipid metabolism has been suggested but this remains to be clearly demonstrated to reside within the lysosome [27,45]. EPDR1 is demonstrated. Interestingly, an apparent lysosomal acid phospholi- named after ependymin which is a predominant glycoprotein in the pase B activity was demonstrated using an electron microscopic cerebrospinal fluid of teleost fishes [72]. Piscine ependymins have approach in liver and kidney from mice [69]. been suggested to be associated with neoplasticity and regeneration The highly glycosylated P67 from Trypanosoma brucei displays of the brain by modulating calcium homeostasis (reviewed in [73]). 28% identity to human P76 but differs in an additional C-terminal Mammalian EPDR1 was first described as a gene that was shown to be transmembrane domain and thus resembles the lysosomal integral upregulated in human colorectal tumor specimens (hence designated membrane proteins LAMP-1/-2 from a structural point of view [70]. “upregulated in colorectal cancer gene-1”) [74] and down-regulated The depletion of P67 in trypanosomes by RNAi knockdown drama- in hematopoetic cells [75]. While EPDR1 is clearly lysosomal, tically alters lysosomal morphology and function and eventually homology searching fails to any significant similarity to other proteins leads to retarded growth and death of the parasite in the mam- of known function that might provide clues to its cellular role. From malian bloodstream [71]. In RNAi-treated trypanosomes of blood- this perspective, EPDR1 represents a particularly interesting new stream origin, lysosomes were extremely enlarged (4–6times)and lysosomal protein that will require further investigation to ascertain exhibited autophagosome-like membranes [71]. The observation its function. that ablation of the trypanosome ortholog of P76 results in a severe lysosomal defect suggests that this protein may well be worth 8.9. Serine carboxypeptidase 1, retinoid-inducible serine further investigation in human lysosomal storage diseases of un- carboxypeptidase (SCPEP1) known etiology. SCPEP1 was originally identified in a screen for genes responding 8.7. FLJ22662 to retinoic acid in rat vascular smooth muscle cells [76].SCPEP1is classified as a member of the serine carboxypeptidase family S10 A second member of the -related family, FLJ22662 which is characterized by a of Ser, Asp and His and is located on 12p13 and encodes a protein that is which exhibit peptidase activity at acidic pH, which is consistent predicted to be 552 amino acids long and which is 32% identical to with lysosomal function [77]. SCPEP1 is 23% identical to the lyso- P76. Like P76, a potential catalytic function for this protein remains to somal serine carboxypeptidase protective protein/cathepsin A and be determined. 20% identical to vacuolar carboxypeptidase Y from S. cerevisiae.The 35 kDa mature SCPEP1 arises from a 51 kDa precursor and both a C- 8.8. Mammalian ependymin-related protein 1 (EPDR1) terminally fluorescent-tagged derivative and the endogenous pro- tein have been shown to localize to the lysosome [45,78]. However, First purified from rat brain [35], human and rodent EPDR1 have to date, no enzymatic activity has been demonstrated for SCPEP1 since been found in most proteomic analyses of M6P-glycoproteins. [78]. 632 T. Lübke et al. / Biochimica et Biophysica Acta 1793 (2009) 625–635

8.10. Epididymis-specific alpha-D-mannosidase (MAN2B2) of these proteins may be incorrectly assigned or they may have multiple cellular locations. A number of ER/Golgi proteins are In addition to the well known lysosomal acid alpha-D-mannosidase frequently seen, including procollagen-lysine 1,2-oxoglutarate 5- (MAN2B1), MAN2B2 was purified as a second alpha-D-mannosidase dioxygenase 1 and microsomal stress 70 protein ATPase [5].In in porcine epididymal fluid [79]. The 135 kDa MAN2B2 precursor is addition, numerous abundant plasma proteins have also been found processed into subunits with apparent molecular weights of 63 kDa and surprisingly shown to contain Man6-P [40]. The significance of and 51 kDa, respectively [79]. The mouse ortholog is ubiquitously Man6-P on these proteins is unclear but it is possible that they may expressed [80]. Identified in most studies of purified Man6-P glyco- simply represent low affinity substrates for the lysosomal phospho- proteins, human MAN2B2 has recently been shown to preferentially that receive the Man6-P modification to some degree. For cleave the core alpha 1,6-mannose residue from the Man3-GlcNAc but ER/Golgi proteins, this may simply reflect spatial proximity to the not from Man3-GlcNAc2 or larger high mannose oligosaccharides [81]. phosphotransferase. For abundant plasma proteins, this may reflect A lysosomal alpha-mannosidase specific for alpha 1,6-linked core their high abundance within the biosynthetic secretory pathway mannose residues, probably representing MAN2B2, was partially combined with the fact that they are low affinity ligands for the purified from human alpha-mannosidosis fibroblasts, human spleen lysosomal enzyme phosphotransferase, resulting in a proportion of and rat liver [82–84]. each receiving the Man6-P modification. Thus, while a lysosomal function for some of these glycoproteins cannot be ruled out, further 8.11. 1, 6 and T2 (RNASE1, RNASE6, RNASET2) studies are needed to understand the significance of Man6-phosphor- ylation in these cases. Three ribonucleases have been purified by MPR affinity chromato- graphy including (RNASE1), ribonuclease 6 9. Novel lysosomal membrane proteins (RNASE6) and ribonuclease T2 (RNASET2) and these are promising lysosomal candidates due to their respective hydrolytic activities. For Subcellular fractionation, genetic and other approaches have RNASET2, Man6-phosphorylation has been experimentally verified recently led to the discovery of several new lysosomal membrane [37] and a lysosomal localization was recently demonstrated by proteins and provided many candidates for localization to this subcellular fractionation [85]. compartment (summarized in Table 3).

8.12. Cat eye syndrome critical region 1 (CECR1) 9.1. Lysosomal p40 (C2orf18)

CECR1 is a human homolog of insect-derived growth factor and Lysosomal p40 (gene name 4930471M23Rik) was originally encodes an adenosine deaminase isoenzyme (ADA2) that was found identified in a preparation of lysosomal membranes derived from rat at low levels in human serum and may be active at sites of liver lysosomes [90]. Lysosomal localization was subsequently demon- inflammation during hypoxia and of tumor growth [86]. Interestingly, strated by cosedimentation with lysosomal marker proteins after the pH optimum for adenosine deaminase activity is slightly acidic subcellular fraction and colocalization of GFP-tagged versions of p40 (∼pH 6.6) which could reflect a lysosomal function. with LAMP-1 [90]. Mouse p40 is a highly hydrophobic integral membrane protein that has between seven to ten transmembrane 8.13. Acyloxyacyl hydrolase (AOAH) domains that are linked to each other by relatively short loops. Unlike most lysosomal membrane proteins, p40 does not contain any N- AOAH specifically deacylates bacterial lipopolysaccharide endo- linked carbohydrate residues. The lysosomal half-life of p40 was toxins and in doing so, is thought to play an important role in host estimated to be ∼10 h [90] which is comparable with highly- defence. This protein has some sequence similarity with lysosomal glycosylated proteins such as LAMP-1 or LIMP-1, -2 or -3 for which saposins, has been experimentally determined to contain Man6-P, can deglycosylation results in an increased [91,92]. Sequence be endocytosed in an MPR-dependent manner [87], has a pH optima homologies suggest that p40 may function as a lysosomal - for catalytic activity between 5 and 6 depending on [88] and, sugar transporter. when expressed in BHK cells, it is present in vesicular cytoplasmic bodies [89]. All of these observations are consistent with a lysosomal 9.2. Transmembrane protein 74 (TMEM74) localization. A number of proteins that are routinely identified in MPR-affinity Initially, TMEM74 was identified in a genetic screen for human purified preparations are not currently assigned to the lysosome but ORFs that could promote autophagy in cultured cells [93]. TMEM74 is are assigned to cellular compartments or thought to be secreted. Some a 305 amino acid protein with a predicted molecular weight of

Table 3 Novel lysosomal membrane proteins

Membrane proteins Gene Ref. Putative sorting signals (exxxll-like, yxxΦ-like) Lysosomal validation Lysosomal p40 C2orf18 a, e Yes Morphological, subcellular fractionation Transmembrane protein 74 TMEM74 f Yes, depending on orientation Morphological Heparan-α-glucosaminide N-acetyl-transferase (TMEM76) HGSNAT b, c, e Yes Morphological Major facilitator superfamily domain containing 8 protein MFSD8 d, e Yes Morphological (MFSD8 alias CLN7) LOC201931 alias FLJ38482 TMEM192 e Yes Morphological C7orf28A alias LOC51622 C7orf28A e Yes, depending on orientation Morphological

References are: a, Boonen et al. [90], Biochem J 395, 39–47; b, Fan et al. [20], Am J Hum Genet 79, 738–744; c, Hrebicek et al. [96], Am J Hum Genet 79, 807–819; d, Siintola et al. [98], Am J Hum Genet 81, 136–146; e, Schröder et al. [47], Traffic 8, 1676–1686; and f, Yu et al. [93], Biochem Biophys Res Commun 369, 622–629. Φ, large hydrophobic amino acid. T. Lübke et al. / Biochimica et Biophysica Acta 1793 (2009) 625–635 633

33.3 kDa and is conserved in . It is of unknown function and of potential novel candidates for residence in this organelle. The list of lacks homology to other proteins that may provide clues to its potentially new lysosomal proteins is likely to continue to grow with the molecular role. However, TMEM74-GFP fusion protein was shown to discovery of other proteins that are restricted to particular cells or tissues localize to lysosomes and autophagosomes and it appears important that have yet to be analyzed using proteomic approaches. This is in the regulation of autophagy [93] as its ablation by RNAi approaches illustrated with a recent analysis of multiple rat tissues [4] that has strongly inhibited starvation-induced autophagy. yielded even more potential lysosomal candidates. In addition, improve- ments in the sensitivity and accuracy of mass spectrometric methods for 9.3. Heparan-alpha-glucosaminide N-acetyltransferase protein identification will increasingly facilitate the identification of low (HGSNAT, TMEM76) abundance constituents of mixtures of lysosomal proteins. Despite considerable progress, there remain significant obstacles to Heparan alpha-glucosaminide N-acetyltransferase activity has long a complete characterization of the composition and function of the been associated with the lysosomal compartment and with the lysosome using proteomic approaches. First, as methods for protein lysosomal storage disorder mucopolysaccharidosis IIIC (MPS IIIC, or identification become more sensitive, contaminants, as well as bona Sanfilippo C) [94]. Prior to its discovery, a transmembrane acetylation fide lysosomal proteins, will increasingly be identified, potentially model was hypothesized in which HGSNAT was proposed to be a resulting in more false positive assignments to the lysosome. To a membrane protein that transfers acetyl-residues from cytosolic acetyl- degree, the problems associated with differentiating between proteins CoA onto luminal heparan sulfate [95]. Despite these insights into the of interest and contaminants is countered by an increasing general mechanism of HGSNAT action, its gene was not identified until 2006, awareness of the need for parallel control samples in proteomic studies when two groups reported its discovery using different approaches. One together with robust biostatistical methods for data analysis [49]. study used a traditional genetic linkage analysis of 27 patients and 17 Second, in validating candidates identified by both subcellular unaffected relatives [96] while the other used a proteomic candidate fractionation and MPR-affinity chromatography, it is likely that in approach (see above) [20]. HGSNAT consists of 635 amino acids with 11 future it may be insufficient to simply show that a given protein can predicted transmembrane domains and several tyrosine-based and di- localize to the lysosome as this will not differentiate functional leucine motifs in its cytosolic C-terminus for lysosomal targeting. The lysosomal components from proteins undergoing lysosomal degrada- lysosomal localisation of HGSNAT was confirmed by immunofluores- tion or the small proportion of some proteins that may aberrantly cence studies and wild type HGSNAT cDNA was shown to functionally receive the Man6-P modification and be targeted to and stable in the reconstitute N-acetyltransferase activity in fibroblasts of MPS IIIC lysosome. It is thus becoming clear that, in the validation of lysosomal patients. Subsequent studies of MPS IIIC patients have revealed a candidates, it is necessary to not only demonstrate a lysosomal range of mutations in HGSNAT [20,96,97]. localization but also determine the proportion of the endogenous protein that resides within this compartment. Thirdly, many of the new 9.4. Major facilitator superfamily domain containing 8 protein lysosomal proteins that are beginning to emerge are of completely (MFSD8, CLN7) unknown function, and while knowing that they are lysosomal is valuable in its own right, a lack of functional information limits our Mutations in the MFSD8 gene are the molecular cause of variant understanding of the global role of the lysosomal system. In many late-infantile-onset neuronal ceroid lipofuscinosis (LINCL) in a cases, as described earlier, homology with proteins of known subset of Turkish LINCL patients [98].MFSD8encodesahighly function can provide useful clues, but this is not always the case conserved 518 amino acids protein with a calculated molecular (e.g. EPDR1). Understanding the role of such proteins is of particular weight of ∼58 kDa and 12 predicted transmembrane domains. interest as they could potentially facilitate completely new lysosomal Expression of a tagged version of MFSD8 in COS-1 and HeLa cells functions. 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